Inhibition of SARS coronavirus infection with clinically approved antiviral drugs

ABSTRACT

The invention relates to methods for treating or inhibiting SARS-CoV infection involving the administration of an interferon, particularly IFN α-n1, IFN α-n3, human leukocyte IFN α or IFN β-1b.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/476,611, filed Jun. 9, 2003, which is herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates generally to treatment of viral infection,and particularly to SARS-CoV infection.

BACKGROUND OF THE INVENTION

Severe acute respiratory syndrome (SARS) (1,2) is an infectious diseasecaused by a newly identified human coronavirus (SARS-CoV) (3,4).SARS-CoV has a single-stranded, positive-sense RNA genome of 30 kb, andencodes 14 potential open reading frames (ORFs).

Symptoms of SARS includes fever over 38° C. and other symptoms such asheadache, an overall feeling of discomfort, body aches and mildrespiratory symptoms at the outset. About 10 percent to 20 percent ofpatients have diarrhea After 2 to 7 days, SARS patients may develop adry cough. Most patients develop pneumonia SARS is a highly contagiousinfectious disease and has a mortality rate of 15 to 20%. Currently, noeffective drug exists to treat SARS-CoV infection (5).

Intensive efforts are under way to gain more insight into the mechanismsof SARS-CoV replication, in order to develop targeted antiviraltherapies and vaccines. Developing effective and safe vaccines andchemotherapeutic agents against SARS-CoV, however, may take years. Therecent epidemic has shown that knowledge is lacking regarding theclinical management and treatment of infected patients. Ribavirin(6-12), oseltamivir (8-10), foscarnet (8), intravenous immunoglobulin(8), and other agents have been used to treat patients. Preliminaryresults from in vitro testing indicate that ribavirin concentrationsthat inhibit other viruses sensitive to ribavirin do not inhibitreplication or cell-to-cell spread of the SARS-CoV (5). However, theU.S. Centers for Disease Control and Prevention concluded that furtherin vitro testing of antiviral drugs on other coronavirus isolates andmore information on the clinical outcome of patients treated withribavirin or other antiviral drugs in controlled trials is needed (5).

SUMMARY OF THE INVENTION

The present invention relates to methods and uses of various interferonsto inhibit SARS-CoV infection. Thus, in one aspect, the presentinvention provides a method of treating SARS-coronavirus infection,comprising administering an effective amount of an interferon to apatient, wherein the interferon is IFN α-n1, IFN α-n3, human leukocyteIFN α or IFN β-1b. The invention also provides use of an effectiveamount of an interferon for treating SARS-coronavirus infection, and useof an effective amount of an interferon in the manufacture of amedicament for treating SARS-coronavirus infection, wherein theinterferon is IFN α-n1, IFN α-n3, human leukocyte IFN α or IFN β-1b.

In another aspect, the invention provides a method of inhibitingSARS-coronavirus infection, the method comprising administering aninterferon to a cell capable of being infected with SARS-CoV, whereinthe interferon is IFN α-n1, IFN α-n3, human leukocyte IFN α or IFN β-1b.The invention also provides use of an interferon for inhibitingSARS-coronavirus infection, and use of an interferon in the manufactureof a medicament for inhibiting SARS-coronavirus infection, wherein theinterferon is IFN α-n1, IFN α-n3, human leukocyte IFN α or IFN β-1b.

The present invention also provides kits for use in practicing themethods of the invention. In various aspects, there is provided a kitcomprising an interferon and instructions for using the interferon totreat SARS-coronavirus infection, or to inhibit SARS-coronavirus,wherein the interferon is IFN α-n1, IFN α-n3, human leukocyte IFN α orIFN β-1b.

Other aspects and features of the present invention will become apparentto those of ordinary skill in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, which illustrate, by way of example only, embodiments ofthe present invention,

FIG. 1 is a photograph of VeroE6 cells either (a) uninfected or (b)infected SARS-CoV, demonstrating the cytopathic effects of SARS-CoV;

FIG. 2 is a line graph illustrating a dose response curve for interferonα-n3 as determined by a plaque reduction assay;

FIG. 3 is a line graph illustrating a dose response curve for interferonβ-1b as determined by a plaque reduction assay; and

FIG. 4 is a line graph illustrating a dose response curve for humanleukocyte interferon α as determined by a plaque reduction assay.

DETAILED DESCRIPTION OF EMBEDMENTS

The present invention relates to the discovery that certain interferon(IFN) subtypes inhibit infection by SARS coronavirus (SARS Co-V).

Interferons are a class of proteins produced by a host organism tocombat viral infection and tumour development. Host cells, when infectedby a virus, synthesize and secrete interferon, which then binds toreceptors on healthy cells and induces an antiviral response mechanismin such cells. The biologic response properties of interferons aremediated through its interactions with specific surface cell receptors,leading to activation of the JAK/STAT pathway and productioninterferon-stimulated gene products such as 2′5′-oligoadenylate synthaseand protein kinase PKR (24). The antiviral activity of interferons ismediated by direct effects on infected cells or through an inducedimmune response (23).

The inventors have discovered that certain interferon subtypes,particularly IFN α-n1, IFN α-n3, human leukocyte IFN α and IFN β-1b,inhibit SARS-CoV infection in Vero E6 cells in vitro. The resultsindicate that various IFNs can be used to inhibit SARS-CoV infection,and therefore are useful in the treatment of individuals infected withSARS-CoV.

The invention therefore provides a method of treating SARS-CoV infectionby administering an effective amount of an interferon to a patient. Theinvention also provides a method of inhibiting SARS-CoV infection,comprising administering an interferon to a cell capable of beinginfected with SARS-CoV.

“Treating” SARS-CoV infection refers to an approach for obtainingbeneficial or desired results, including, but not limited to,eradication, alleviation or amelioration of infection, diminishment ofextent of infection and prevention or slowing of progression or spreadof infection. As will be understood by a skilled person, results may notbe beneficial or desirable if, while improving a state of infection in apatient, the treatment results in adverse effects on the patient treatedwhich outweigh any benefits effected by the treatment.

The interferon is an interferon that is capable of inhibiting SARS-CoVinfection. Inhibiting SARS-CoV or inhibiting SARS-CoV infection refersto the ability of a particular interferon to prevent or reduce invasionby SARS-CoV into cells to thereby cause SARS, either in a patient or invitro, or to prevent or reduce SARS-CoV from spreading from infectedcells to uninfected cells to thereby prolong an existing infection,either within an infected individual or in vitro, or to prevent SARS-CoVfrom replicating within an infected cell. The interferon as that term isused herein includes a full-length interferon and a fragment of afull-length interferon that maintains the inhibitory activity of thefull-length interferon. A skilled person will be able to determinewhether a fragment of a full-length interferon is capable of maintainingthe inhibitory activity, including an antiviral response, in a targetcell, for example as described in the Examples.

The interferon may be an interferon isolated and purified from a cellpopulation that normally produces interferon, for example leukocytes orlymphoblastoid cells. Alternatively, the interferon may be a recombinantinterferon, produced by the cloning and expression of an interferon genein an expression system, for example, recombinantly produced in E. colior mammalian cell culture, and subsequent purification of the expressedinterferon. Recombinant protein expression techniques are known in theart, and described for example in Sambrook et al. ((2001) MolecularCloning: a Laboratory Manual, 3^(rd) ed., Cold Spring Harbour LaboratoryPress). Interferons that are approved for clinical use are commerciallyavailable, including recombinant interferons and interferons isolatedfrom a natural source. Such commercially available interferons includeRoferon™ (Roche), Intron A™ (Schering-Plough), Wellferon™(Glazosmithkline), Alferon™ (Hemispheryx), Rebif™ (Serono), Betaferon™(Schering AG) and Multiferon™ (Viragen International Inc).

In certain embodiments, the interferon is IFN α-n1, IFN α-n3, humanleukocyte IFN α or IFN β-1b.

The cell may be one or more cells capable of being infected withSARS-CoV, meaning SARS-CoV is able to infect such cells, and includes acell that is already infected with SARS-CoV. Such a cell or cells May bein a patient, or may be a cell in vitro, for example, an isolated cell,or a cell that is part of a cell population or cell culture. Cells thatare capable of being infected by SARS-CoV include epithelial cells,blood cells, including macrophages, and cells of heart, liver, kidneys,or eyes. Identification of cells that are capable of being infected withSARS-CoV may be achieved by growing cells in vitro, and performing astandard plaque assay using SARS-CoV. Alteratively, cell samples may betaken from an individual known to be, or suspected of being, infectedwith SARS-CoV, and tested for the presence of SARS-CoV, for example byusing PCR amplification methods to detect the presence of the viralgenome in particular cell types isolated from the patient.

The cell may be a human cell, for example, peripheral blood leukocytesor a THP-1 monocytic cell, or it may be derived from another species,where that cell is capable of being infected by SARS-CoV, for example aVERO 6 cell.

When the cell is an in vitro cell, administering may be achieved byadding the interferon to the growth medium, either prior to exposure ofthe cells to SARS-CoV so as to prevent infection of the cells by thevirus, or after to exposure to SARS-CoV, or concomitantly with SARS-CoV.

The patient may be any animal, including a human. In a particularembodiment, the patient is a human.

The interferon may be administered to a patient using standard methodsof administration. In various embodiments, the interferon isadministered systemically, including orally, parenterally or by anystandard method known in the art.

When administered to a patient, an effective amount of the interferon isthe amount required, at the dosages and for sufficient time period, toameliorate, to alleviate, improve, mitigate, ameliorate, stabilize,prevent the spread of, slow or delay the progression of or cure theinfection. For example, it may be an amount sufficient to achieve theeffect of reducing or eliminating viral load, or the effect of reducingor stabilizing the number of cells infected with SARS-CoV, or inhibitingthe replication and/or proliferation of SARS-CoV, or preventing orslowing the spread of virus from infected to uninfected cells.

The effective amount to be administered to a patient can vary dependingon many factors such as the pharmacodynamic properties of theinterferon, the mode of administration, the age, health and weight ofthe patient, the nature and extent of the disease state, the frequencyof the treatment and the type of concurrent treatment, if any, and theprotein activity of the interferon preparation.

One of skill in the art can determine the appropriate amount based onthe above factors. The interferon may be administered initially in asuitable amount that may be adjusted as required, depending on theclinical response of the patient. The effective amount of interferon canbe determined empirically and depend on the maximal amount of theinterferon that can be administered safely, and the minimal amount ofthe interferon that produces the desired result.

The concentration of interferon to be administered will vary dependingon the ability of the particular type or subtype of interferon that isto be administered, as well as the source of interferon and amount ofglycosylation. For example, for certain subtypes of IFN α, the amountmay vary from about 3-5 million IU three times a week to about 5 millionIU daily for administration into a human patient. For IFN β subtypes,doses of about 3-6 million IU may be administered three times weekly.Plasma levels of interferons administered via the subcutaneous route areusually low with correspondingly short half-lives. In view of theirmechanism of action, absolute serum levels may not be meaningful as ameasure of the biological activity of interferons, compared to inductionof cellular products such as 2′5′-oligoadenylate synthase.

Effective amounts of interferon can be given repeatedly, depending uponthe effect of the initial treatment regimen. Administrations aretypically given periodically, while monitoring any response. It will berecognized by a skilled person that lower or higher dosages than thoseindicated above may be given, according to the administration schedulesand routes selected.

The interferon may be administered alone or in combination with anadditional antiviral agent. The additional antiviral agent may be anyagent that exhibits an antiviral effect when administered to anindividual infected with SARS-CoV. For example the other antiviral agentmay be a nucleoside analogue, a protease inhibitor, a reversetranscriptase inhibitor, a neuraminidase inhibitor, or a receptorantagonist.

For example, in one embodiment, tie interferon is administered incombination with ribavirin. Ribavirin is a nucleoside analogue, and asset out in the Examples below, inhibitory activity was observed at allviral loads tested, albeit with relatively high concentrations ofribavirin (0.5 to 5 mg/mL). However, concentrations of ribavirinrequired to demonstrate an inhibitory effect on SARS-CoV resulted inobserved slight cytotoxicity. These results indicate that althoughribavirin is not suitable for treatment of SARS-CoV infection alone, itmay be used in combination with other treatments at lower doses. Thus,in certain embodiments, ribavirin may be administered in combinationwith the interferon. In some embodiments, the dose of ribavirinadministered to a human subject in combination with the interferon isbetween about 600 and about 1200 mg per day, which dose has been shownto be effective as a combination therapy against hepatitis C infection,see for example U.S. Pat. No. 6,685,931, which is herein incorporated byreference.

In combination with refers to concurrent or sequential administration ofinterferon and an additional antiviral agent. When administeredconcurrently, the interferon and additional antiviral agent may beadministered together, in the same vehicle or dosage form, or may beadministered in separate vehicles or dosage forms, although administeredat the same time. When administered sequentially, the administration ofthe interferon, if given in multiple doses, may overlap with the timingof the administration of one or more dose of the additional antiviralagent, such that the two are administered within the course of a commontreatment schedule to achieve the desired combined treatment effect. Theroutes of administration of the interferon and the additional antiviralagent, whether administered concurrently in different vehicles or dosageforms, or administered sequentially, may be the same or different.

The interferon may be administered alone or in combination with apharmaceutically acceptable carrier, the proportion of which isdetermined by the solubility and chemical nature of the interferon,chosen route of administration and standard pharmaceutical practice.

To ease administration, the interferon may be formulated as aningredient in a pharmaceutical composition. Therefore, in oneembodiment, there is provided a pharmaceutical composition comprising aninterferon and a pharmaceutically acceptable carrier, in a biologicallycompatible form suitable for administration in vivo.

The pharmaceutical composition may further comprise an additional viralagent, as described above, including ribavirin.

The pharmaceutical compositions may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives andvarious compatible carriers. For all forms of delivery, the recombinantvirus may be formulated in a physiological salt solution.

The proportion and identity of the pharmaceutically acceptable carrieris determined by chosen route of administration, compatibility with anactive protein and standard pharmaceutical practice. Generally, thepharmaceutical composition will be formulated with components that willnot significantly impair the biological activity of the interferon.

The pharmaceutical composition can be prepared by known methods for thepreparation of pharmaceutically acceptable compositions suitable foradministration to patients, such that an effective quantity of theactive substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985). On thisbasis, the compositions include, albeit not exclusively, solutions ofthe interferon in association with one or more pharmaceuticallyacceptable vehicles or carriers, and contained in buffer solutionshaving a suitable pH and which are iso-osmotic with physiologicalfluids.

The pharmaceutical composition may be administered to a patient in avariety of forms depending on the selected route of administration, aswill be understood by those skilled in the art. The composition of theinvention may be administered orally or parenterally. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous,intramuscular, transepithelial, transdermal, nasal, intrapulmonary,intrathecal, rectal and topical modes of administration. Parenteraladministration may be by continuous infusion over a selected period oftime.

The pharmaceutical composition may be administered orally, for example,with an inert diluent or with an assimilable carrier, or it may beenclosed in hard or soft shell gelatin capsules, or it may be compressedinto tablets or it may be incorporated directly with the food of thediet. For oral therapeutic administration, the interferon may beincorporated with an excipient and be used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers and the like.

The pharmaceutical compositions may also be administered parenterally orintraperitoneally. Solutions of the interferon may be prepared in aphysiologically suitable buffer. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms, but that will not inactivate the interferon protein.A person skilled in the art would know how to prepare suitableformulations. Conventional procedures and ingredients for the selectionand preparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences and in The United StatesPharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

The forms of the pharmaceutical composition suitable for injectable useinclude sterile aqueous solutions or dispersion and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists.

The dose of the pharmaceutical composition that is to be used depends onthe severity and stage of the infection, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent antiviral therapy(if any), the specific/route of administration and other similar factorsthat are within the knowledge and expertise of the health practioner.These factors are known to those of skill in the art and can beaddressed with minimal routine experimentation.

The interferon, or pharmaceutical compositions comprising theinterferon, may be packaged as a kit and the invention in one aspectprovides a kit comprising an interferon, and instructions for use of theinterferon to treat SARS-CoV infection, or instructions for use of theinterferon to inhibit SARS-CoV. In one embodiment, the kit may furthercomprise an additional antiviral agent, for example ribavirin, andinstructions for concurrent or sequential administration of theinterferon and additional antiviral agent,

The present invention also contemplates various uses of an interferon,including the use of an interferon for treating SARS-CoV infection, useof an interferon in the manufacture of a medicament for treatingSARS-CoV infection, use of an interferon for inhibiting SARS-CoVinfection, and use of an interferon in the manufacture of a medicamentfor inhibiting SARS-CoV infection.

All references referred to herein are incorporated by reference.

EXAMPLES

The aim of this study was to investigate whether a panel of currentlyavailable antiviral agents exhibit in vitro anti-SARS-CoV activity. Acell-based assay utilizing cytopathic endpoints (CPE) was set up usingVero E6 cells to screen these antiviral compounds. SARS-CoV has beenshown to infect Vero E6 cells, an African green monkey kidney cell line(3). It is also contemplated that the following method can be used inconjunction with peripheral blood leukocytes and THP-1 monocytic cellline. The initial screen was followed by a plaque reduction assay todetermine the 50% effective concentration (EC₅₀) of compounds showingpositive results. These experiments allow rapid screening ofcommercially available antiviral agents, enabling those with in vitroevidence of activity to move expeditiously into clinical studies, sincesafety and pharmacokinetic information in humans is already availablefor other disease indications. A total of 19 drugs approved for clinicaluse in the treatment of viral infections were tested, which arerepresentative compounds from major antiviral pharmacologic classes thatare currently commercially available: nucleoside analogues, interferons,protease inhibitors, reverse transcriptase inhibitors and neuraminidaseinhibitors.

Materials and Methods

Selection and Preparation of Drugs: To rapidly identify a pharmacologicagent that could be used to treat SARS, a collection of antiviral drugswas tested against SARS-CoV, the etiologic agent of the atypicalpneumonia To investigate a wide spectrum of potential molecular targets,we decided to cover the entire pharmacologic range of commerciallyavailable antiviral agents, including agents not expected to be activeagainst coronaviruses. Information on antiviral drugs provided here wasobtained from prescribing information sheets or from communications withthe manufacturer.

Nucleoside analogues are a diverse class of compounds; in general, theyinhibit viral RNA or DNA polymerases or other enzymes, interfering withnucleic acid synthesis. In this study, the selected compounds thattarget DNA viruses such as herpes simplex virus (HSV) andvaricell-zoster viruses (VZV) were acyclovir, ganciclovir, andfoscarnet. Ribavirin has activity against a range of DNA and RNAviruses; in different cell lines, ED₅₀ ranges from 1 to 100 μg/mL.Antiretroviral (HIV) drugs include reverse transcriptase (RT) inhibitorsand protease inhibitors. Selected HV nucleoside RT inhibitors studiedwere zidovudine and lamivudine, while HIV protease inhibitors studiedwere indinavir, nelfinavir, and saquinavir. The third group ofantivirals studied were the neuraminidase inhibitors; both commerciallyavailable preparations, zanamivir and oseltamivir, were used in thisstudy. Interferons were the next major class of antivirals studied.Various subtypes of interferon α (2a, 2b, n1, and n3, human leukocyte)and β (1a and 1b) were used. Amantadine, an old antiviral compound, wasalso studied. Different terms have been used to express antiviralactivity, namely, 50% effective concentration (EC₅₀), 95% effectiveconcentration (EC₉₅), 50% effective dose (ED₅₀), 50% inhibitoryconcentration (IC₅₀) 95% inhibitory concentration (IC₉₅), and minimuminhibitory concentration (MIC). Table 1 illustrates the range ofactivity for particular antiviral drugs against certain viruses.

TABLE 1 Inhibitory Concentrations of Antiviral Drugs against SelectViruses Compound IC₅₀ Virus Foscavir 50-800 μmol/L Cytomegalovirus 5-443μmol/L Herpes simplex mutants Acyclovir 0.01-13.5 μg/mL Herpes simplexvirus and varicella-zoster virus Cymevene 0.02-3.48 μg/mL Laboratorystrains or clinical isolates of cytomegalovirus Ribavirin 1-25 μg/mLInfluenza 25-100 μg/mL HIV and other retro- viruses 3.2-50 μg/mL (MIC)Herpes and poxviruses suppression Lamivudine 0.0006-0.034 μg/mL HIVZidovudine 0.003-0.013 μg/mL HIV Fortovase 1-30 nmol/L HIV Viracept7-196 nM (EC₉₅) HIV Crixivan 25-100 nmol/L HIV Relenza 0.005-16 μmolInfluenza virus Tamiflu 0.0008 μM→35 μmol Influenza virus Amantadine0.1-25 (ED₅₀) Influenza virus ^(a)IC₅₀, 50% inhibitory concentration;EC₉₅, 95% effective concentration; ED₅₀, 50% effective dose.

Tenfold dilutions of the drug were tested to cover a broad range ofconcentrations above and below inhibitory dosages as reported by themanufacturer for other viral-host combinations. Compounds alreadypresent in aqueous injections were made up to volume by using Hank'sbuffered saline solution. For tablet and capsule formulations withsoluble active ingredients, the outer coat was removed whereverapplicable, and the preparation was ground in a mortar and pestle. Thecontents were dissolved in water, vortexed, and centrifuged thereafterat 3,000 g. The required volume was pipetted from the supernatant anddiluted accordingly. When the active ingredients were insoluble in water(nelfinavir and saquinavir), the contents were dissolved indimethylsulphoxide (DMSO); care was taken to ensure that the finalconcentration of DMSO in the dilutions would not exceed 1%. For plaqueassays, fivefold drug dilutions were prepared by using growth media asspecified below.

SARS-CoV Production and Infection: Vero E6 cells (American Type CultureCollection, Manassas, Va.) were propagated in 75 cm 2 cell cultureflasks in growth medium consisting of medium 199 (Sigma, St Louis, Mo.)supplemented with 10% fetal calf serum (FCS; Biological Industries,Kibbutz Beit Haemek, Israel). SARS-CoV 2003VA2774 (an isolate from aSARS patient in Singapore), which has been previously sequenced (14),was propagated in Vero E6 cells. Briefly, 2 mL of stock virus was addedto a confluent monolayer of Vero E6 cells and incubated at 37° C. in 5%CO₂ for 1 h; 13 mL of medium 199 supplemented with 5% FCS was thenadded. The cultures were incubated at 37° C. in 5% CO₂, and thesupernatant was harvested after 48 h; in >75% of cultures, inhibition ofCPE (3+) in each well was observed with an inverted microscope. Thesupernatant was clarified at 2,500 rpm and then divided into aliquots,placed in cryovials, and stored at −80° C. until use.

Virus Handling and Titration: All virus culture and assays were carriedout in the biosafety Level 3 laboratory at the Environmental HealthInstitute, according to the conditions set out in Biosafety inMicrobiological and Biomedical Laboratories (15). Virus titer in thefrozen culture supernatant was determined by using a plaque assay.Briefly, 100 μL of virus in 10-fold serial dilution was added, induplicates, to a monolayer of Vero E6 cells in a 24-well plate. After 1h of incubation at 37° C. in 5% CO₂, the viral inoculum was aspirated,and 1 mL of carboxymethylcellulose overlay with medium 199, supplementedwith 5% FCS, was added to each well. After 4 days of incubation, thecells were fixed with 10% formalin and stained with 2% crystal violet.The plaques were counted visually, and the virus titer in plaque-formingunits per mL (PFU/mL) was calculated.

Cytopathic Endpoint Assay: The protocol used was adapted from Al-Jabriet al. (16), and all drugs were tested in quadruplicate. Briefly, 100 μLof serial 10-fold dilutions of the drugs were incubated with 100 μL ofVero E6 cells, giving a final cell count of 20,000 cells per well in a96-well plate. The incubation period was 1 h at 37° C. in 5% CO₂, exceptfor the interferons, which were incubated overnight with the cells. Tenmicrolitres of virus at a concentration of 10,000 PFU/well was thenadded to each of the test wells. The plates were incubated at 37° C. in5% CO₂ for 3 days and observed daily for CPE. The end point was the drugdilution that inhibited 100% of the CPE (CIA₁₀₀) in quadruplicate wells.To determine cytotoxicity, 100 μL of serial 10-fold dilutions of thedrugs was incubated with 100 μL of Vero E6 cells, giving a final cellcount of 20,000 cells per well in a 96-well plate, without viralchallenge. The plates were then incubated at 37° C. in 5% CO₂ for 3 daysand examined for toxicity effects by using an inverted microscope.

Plaque Reduction Assay: Trypsinized Vero E6 cells were resuspended ingrowth medium and preincubated with interferons (serial fivefolddilution) in quadruplicate wells in 24-well plates. The next day, themedium was aspirated, and 100 μL of virus was added to each well at atiter of 100 PFU/well. After incubation for 1 h, the virus inoculum wasaspirated, and a carboxymethylcellulose overlay containing maintenancemedium and the appropriate interferon concentration was added. After 4days' incubation, the plates were fixed and stained as describedpreviously. The number of plaques was then counted visually, and theconcentration of drug that inhibits 50% of plaques in each well (IC₅₀)was determined. Results were plotted in Microsoft Excel, and apolynomial of order three was used to approximate the data andextrapolate IC₅₀ and IC₉₅ values.

Results

Cell-based Assay of SARS-CoV Infection: High titers of infectiousSARS-CoV, originally derived from a respiratory sample of a SARSpatient, were propagated on Vero E6 cells. The CPE of SARS-CoV on VeroE6 was evident within 24 hours after infection (FIG. 1).SARS-CoV-infected cells display a CPE characterized by the appearance ofrounded cells and the destruction of the monolayer.

Antiviral Drug Activity: A collection of 19 antiviral drugs was testedin the SARS-CoV CPE inhibition assay (Table 2). The set of drugs testedincluded seven interferons, five nucleoside analogues, three proteaseinhibitors, two RT inhibitors, and two neuraminidase inhibitors.

TABLE 2 Various Antiviral Drugs Tested for Inhibitory Effect on SARS-CoVInhibition Highest of Concentration Cytopathic Antiviral Drug SourceTested Effect Interferons Interferon α2a Roche 100,000 IU/mL No(Roferon ™) Interferon α2b Schering Plough 500,000 IU/mL No (Intron A ™)Interferon αn1 Glaxosmithkline 500,000 IU/mL Yes (Wellferon ™)Interferon αn3 Hemispheryx 10,000 IU/mL Yes (Alferon ™) Interferon β1aSerono 500,000 IU/mL No (Rebif ™) Interferon β1b Schering AG 100,000IU/mL Yes (Betaferon ™) Human Leukocyte Viragen 500,000 IU/mL YesInterferon α International Inc (Multiferon ™) Nucleoside AnaloguesAcyclovir Faulding 1,000 μg/mL No Cymevene Roche 50,000 μg/mL No(Ganciclovir ™) Ribavirin ICN Pharma 10,000 μg/mL Yes FoscarnetAstrazeneca 8,000 μmol/L No (Foscavir ™) Protease Inhibitors Indinavir(Crixivan ™) Merck 100 μmol/L No Nelfinavir Roche 10,000 nmol/L No(Viracept ™) Saquinavir Roche 10,000 nmol/L No (Fortovase ™) ReverseTranscriptase Inhibitors Lamivudine Glaxosmithkline 1,000 μmol/L No(Epivir ™) Zidovudine Glaxosmithkline 1,000 μg/mL No (Retrovir ™)Neuraminidase Inhibitors Oseltamivir Roche 10,000 μmol/L No (Tamiflu ™)Zanamivir Glaxosmithkline 1,000 μmol/L No (Relenza ™) Ion ChannelBlockers Amantadine Novartis 1,000 μg/mL No (Symmetrel ™)

Complete inhibition of the CPE was observed for four of the seveninterferons in the initial screen when very high viral challenge of 10 4PFU/well and a high multiplicity of infection (MOI=0.5) rate were used.Complete inhibition, expressed as CIA₁₀₀, was observed for interferonβ-1b (Betaferon) at 5,000 IU/mL, interferon α-n3 (Alferon) at 5,000IU/mL, interferon α-n1 (Wellferon) at 250,000 IU/mL, and human leukocyteinterferon α (Multiferon) at 500,000 IU/mL. Ribavirin also completelyinhibited the CPE at 5,000 μg/mL (Table 3). None of the other drugsshowed complete inhibition of CPE, even at the highest concentration ofdrug tested (Table 2). Rebif (IFN-β-1a) showed slight inhibition of CPEat 250,000 IU/mL, but the inhibition was not complete at the screeningvirus load of 10,000 PFU/well. Likewise, Roferon (IFN-α-2a) showedslight, incomplete inhibition at 50,000 IU/mL.

Since the criteria for ascertaining anti-SARS-CoV activity in thisscreen were set at 100% inhibition of CPE, and as high doses ofinterferons may result in severe clinical side effects, we chose toconduct further evaluations only in the interferons that showed completeinhibition from initial screen, namely, Wellferon, Multiferon,Betaferon, and Alferon. Based upon results of the primary screen, thefour active interferons and ribavirin were retested at two lower viralchallenges, 10³ and 10² PFU/well. All four drugs again showed inhibitoryeffect, although the CIA₁₀₀ were dependent on viral loads (Table 3). Atthe lowest viral loan the CIA₁₀₀ were 5 IU/mL for both interferon β-1b(Betaferon) and human leukocyte interferon α (Multiferon); and 50 and250 IU/mL for interferon α-n3 (Alferon) and interferon α-n1(Wellferon),respectively. No cytotoxicity of the interferons was observed at or nearinhibitory concentrations. Ribavirin showed inhibitory activity at allthree viral loads, but only at high concentrations of the drug, 0.5-5mg/mL. At high concentrations of ribavirin (0.2-1 mg/mL) cytotoxiceffects were observed on Vero E6 cells, as has been reported for othercell types (17, 18). As such, we consider ribavirin to be inactive as asole treatment against SARS-CoV.

TABLE 3 Complete inhibition of cytopathic effect (CIA₁₀₀) at varyingvirus titers Virus load Ribavarin Wellferon Betaferon Alferon Multiferon(PFU/well) (μg/mL) (IU/mL) (IU/mL) (IU/mL) (IU/mL) 10,000 10,000 500,00010,000 10,000 500,000 1,000 10,000 5,000 1,000 1,000 50 100 1,000 500 10100 5

A plaque reduction assay format with 100 PFU of SARS-CoV (MOI=0.0005)was conducted to determine the IC₅₀ for Betaferon, Alferon, andMultiferon, the three compounds that showed greatest potency forinhibition of CPE. Additional supply was not available for testinginterferon α-n1(Wellferon), as production of this drug has beendiscontinued. Cells were preincubated for 15 h with fivefold dilutionsof drug. Viral-induced plaques, which developed in 3 days, were countedto determine the inhibitory effect of the drugs at variousconcentrations. All three interferon preparations displayed adose-dependent inhibition of SARS-CoV plaque formation in this assay(FIGS. 2-4). For each of the IFNs tested, the amount required to achieve100% inhibition of the cytopathic effect of SARS-CoV (CIA₁₀₀) variedwith viral load (Table 3). The IC₅₀ and IC₉₅, were determined to be 0.2and 8 IU/mL for Betaferon (FIG. 3), 0.8 and 200 IU/mL for Alferon (FIG.2), and 2 and 44 IU/mL for Multiferon (FIG. 4), respectively.

As can be understood by one skilled in the art, many modifications tothe exemplary embodiments described herein are possible. The invention,rather, is intended to encompass all such modification within its scope,as defined by the claims.

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1. A method of inhibiting SARS-coronavirus infection, comprisingadministering an interferon to an in vitro cell capable of beinginfected with SARS-CoV, wherein the interferon is IFN α-n1, IFN α-n3,human leukocyte IFN α or IFN β-1b.
 2. The method of claim 1 wherein theinterferon is recombinant.
 3. The method of claim 2 wherein theinterferon is recombinantly produced in E. coli.
 4. The method of claim2 wherein the interferon is recombinantly produced in mammalian cells.5. The method of claim 1 wherein the cell is a VERO E6 cell, aperipheral blood leukocyte or a THP-1 monocyte.
 6. The method of claim 1further comprising administering to the cell an additional antiviralagent in combination with the interferon.
 7. The method of claim 6wherein the additional antiviral agent is ribavirin.
 8. A method ofinhibiting SARS-coronavirus infection, comprising administering acombination of recombinant interferon and ribavirin to an in vitro VEROE6 cell, a peripheral blood leukocyte or a THP-1 monocyte, said VERO E6cell, said peripheral blood leukocyte or said THP-1 monocyte capable ofbeing infected with SARS-CoV, wherein the interferon is IFN α-n1, IFNα-n3, human leukocyte IFN α or IFN β-1b.